Practical Manual of Biochemistry Website: www.skyfox.co Email:[email protected]About the authors Dr. Sattanathan, Assistant Professor in Life Science, at MASS College of Arts and Science, Kumbakonam, Thanjavur Dt. Tamilnadu, India, has teaching Biotechnology for the last 5 years. He has also worked in other institutes and he has publihsed a number of scientific papers in national and international journals. Dr. Padmapriya has working in Dharmapuram Gnanambigai Govt. Arts College for Women, Mayiladuthurai. She has been in the Fish Immunology field for the last 10 years. Dr. B. Balamuralikrishnan, Ph.D. is an Assistant Professor in Department of Food Science and Biotechnology, Sejong University, Seoul, South Korea. His area of research focuses multidisciplinary in biological science on Molecular Genetics, Food Microbiology, Animal Science, Nutrition Science and Food Biotechnology, especially Food Resources and Microbiological Science. His interests include synthesis of nanomaterials/bioactive compounds from natural- by products and their applications nutraceutical; isolation, and characterization of probiotic for food/feed supplementation and biological field; interested in Nutrition in Aquaculture. To, his credit, he has participated in various International/symposia/conferences in USA, Canada, Japan, Austria, Italy, Czech Republic, Thailand, South Korea and has published more than seventy five research papers in international journal of repute. He has been serving as Academic Editor in Plos One, Guest Editor in Animals [MDPI] and act as potential reviewer in many high reputed journals. He has been acted Life Member in various scientific societies such as The Korean Society of Food Science and Technology, Poultry Science Association, Korean Society of Animal Science and Technology, Animal Nutrition Society of India (ANSI). Dr. B. Balamuralikrishnan, worked as Post-doctoral researcher in Department of Animal Science, Dankook University, South Korea and Research Assistant in Department of Zoology, Bharathiar University, India. Practical Manual of Biochemistry Dr. G. Sattanathan, Ph.D., Dr. S.S. Padmapriya, Ph.D., Dr. B. Balamuralikrishnan, Ph.D.,
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Dr. Sattanathan, Assistant Professor in Life Science, at MASS College ofArts and Science, Kumbakonam, Thanjavur Dt. Tamilnadu, India, has teachingBiotechnology for the last 5 years. He has also worked in other institutes and hehas publihsed a number of scientific papers in national and international journals.
Dr. Padmapriya has working in Dharmapuram Gnanambigai Govt. ArtsCollege for Women, Mayiladuthurai. She has been in the Fish Immunology fieldfor the last 10 years.
Dr. B. Balamuralikrishnan, Ph.D. is anAssistant Professor in Departmentof Food Science and Biotechnology, Sejong University, Seoul, South Korea. Hisarea of research focuses multidisciplinary in biological science on MolecularGenetics, Food Microbiology, Animal Science, Nutrition Science and FoodBiotechnology, especially Food Resources and Microbiological Science. Hisinterests include synthesis of nanomaterials/bioactive compounds from natural-by products and their applications nutraceutical; isolation, and characterizationof probiotic for food/feed supplementation and biological field; interested inNutrition in Aquaculture. To, his credit, he has participated in variousInternational/symposia/conferences in USA, Canada, Japan, Austria, Italy,Czech Republic, Thailand, South Korea and has published more than seventyfive research papers in international journal of repute. He has been serving asAcademic Editor in Plos One, Guest Editor in Animals [MDPI] and act aspotential reviewer in many high reputed journals. He has been acted LifeMember in various scientific societies such as The Korean Society of FoodScience and Technology, Poultry ScienceAssociation, Korean Society ofAnimalScience and Technology, Animal Nutrition Society of India (ANSI). Dr. B.Balamuralikrishnan, worked as Post-doctoral researcher in Department ofAnimal Science, Dankook University, South Korea and Research Assistant inDepartment of Zoology, Bharathiar University, India.
Practical Manualof Biochemistry
Dr. G. Sattanathan, Ph.D.,Dr. S.S. Padmapriya, Ph.D.,Dr. B. Balamuralikrishnan, Ph.D.,
Preface Biochemistry, a fascinating subject dealing with all the body's functions and reactions. Clinical biochemistry has a tremendous effect on patients' diagnosis and treatment. Medical students should be aware of the procedures, the parameters of diagnosis and their estimations. They should acquire sound knowledge of the diagnostic reports and their consequences that contribute to disease diagnosis and prognosis.
The most rapidly growing subject is biochemistry, which is extensively applicable to molecular understanding of the disease. Estiamations of various biochemical parameters definitely provide an insight into the normal metabolism and its aberrations, which form the basis for medicine, leading to diseases. In relation to health and disease, biochemistry shpuld is encouraged that will make the topic more interesting and fascinating for the students. We hope that this practical biochemistry book will help medical students to think about the different facts found in the body's reactions.
Dear students, let us admit that, given the fact that it is non-clinical, extensive, volatile and there is little in it to arouse any amount of interest in medical graduates, biochemistry is seldom a favorite of medical graduates. We have realized that practice is where they have shown some enthusiasm for biochemistry in our biochemistry teaching to undergraduate medical students.
Our main objective is to make this book as simple and attractive as possible for undergraduate biochemistry, allied biochemistry, medical, non-medical students, which is evident from the book's title. Each practical session has been reorganized to achieve this objective in such a way that it is easy to understand. Wherever possible, the subject is presented in tabular format so that it becomes very concise, and all test results are given color so that one realizes the practice of the simple task of reading the book itself. The basic concepts and values behind each experiment are explained in a simple manner.
Our only real concern is to help you understand the subject in an easy and organized way so that not only in your examinations, but also in your future medical career, this little knowledge comes as a big help.
To make this book a better one, we will be happy to accept constructive criticism and fruitful suggestions.
Dr. K. Saravanan
Principal, MASS College of Arts and Science,
Kumabkonam.
ACKNOWLEDGMENTS
I would like to thank God for enabling me to do this work. I
thank my parents, teachers for molding me to reach this
level. I extend my gratitude to my colleagues for their
support.
CONTENTS
1. Rules and Procedures of General Safety 1
2. Buffer solution preparation And pH measurement 3
3. Introduction of Carbohydrates 8
4. Qualitative Analysis of Carbohydrates 10
5. Introduction of Proteins 21
6. Tests for Proteins 23
7. Introduction of Aminoacids 30
8. Tests on Amino Acids 31
9. Qualitative Tests for Lipids 35
10. Sample Collcetion 41
11. Anticoagulants 48
12. Type of Collection Procedures 53
13. Proximate Analysis 55
14. Estimation of Reducing Sugar by Benedict’s Method 56
15. Estimation of Protein by Lowrey’s Method 59
16. Estimation of Cholesterol by Zak’s Method 61
17. Estimation of DNA by Diphenylamine Method 63
18. Separation of Amino Acids by TLC 65
19. Separation of Sugars by Paper Chromatography 66
20. Estimation of CSF Sugar By Trinders Method 68
21. Estimation of RNA by Orcinol Reaction 69
22. Determination of Reducing Sugars 71
23. Effect of Temperatures on Salivary Amylase 73
24. Effect of pH on Salivary Amylase 75
25. Estimation of Haemoglobin 77
26. Estimation of Hb by Cyanmethemoglobin Method 78
27. Isolation of Chloroplast DNA 80
28. Isolation of Mitochondria 82
29. Identification of Lipids by TLC 84
30. Determination of Starch in Plant Tissues 86
31. Isolation of Casein from Milk 88
32. Determination of The Acid Value of a Fat 89
33. Saponification Value of Fat 90
34. Estimation of Blood Cholesterol 91
35. Isolation of RNA From Yeast 92
36. Estimation of Chlorophyll Concentration 93
37. Determination of Total Erythrocyte Count 95
38. Determination of Total Leucocyte Count 96
39. Determination of Packed Cell Volume (PCV) 97
40. Determination of Mean Corpuscular Volume (MCV) 98
41. Determination of Mean Corpuscular Haemoglobin 99
42. Determination of Mean Corpuscular Haemoglobin
Corpuscular 100
43. Differential Leukocyte Count 101
44. Determination of Serum Amylase 102
45. Estimation of Serum Uric Acid 103
46. Phytochemical Analysis 105
47. References 117
1
1. RULES AND PROCEDURES OF GENERAL SAFETY
1. Prior to attending that laboratory session, the laboratory procedures must be
read.
2. Laboratory smoking, eating and drinking are absolutely prohibited at any
time in the laboratory.
3. Only closed-toe shoes should be worn in the lab. Due to the constant risk of
cuts and infections from broken glass found on the laboratory floors and
the possibility of chemical spills, sandals or open-toed or canvas shoes are
not allowed.
4. Keep your face, nose, eyes, ears and mouth away from your hands and other
objects. In the laboratory, the use of cosmetics in the laboratory is
prohibited.
5. Before and after use, work areas or surfaces must be disinfected.
6. While in the laboratory, laboratory coats must be worn and buttoned.
Outside of the laboratory, laboratory coats should not be worn.
7. When conducting any exercise or procedure in the laboratory, protective
eyewear must be worn.
8. To minimize the fire hazard or contamination of experiments, long hair
should be secured behind your head.
9. Prior to leaving the laboratory, hands must be washed.
10. Coats, books and other paraphernalia, such as purses, briefcases, etc., should
be placed in specified locations when entering the laboratory and never on
bench tops (except for your lab manual).
11. Never mouth-pipet anything (including water). Always use appliances for
pipetting.
12. Label all materials with your name, date and any other information
applicable (e.g., media,organism, etc.).
13. Waste disposal in its proper containers (see Biohazard Waste Disposal
below).
14. Note the hazard code on the bottle when handling chemicals and take the
appropriate precautions indicated.
15. Do not pour down the sink with chemicals.
16. Return to their appropriate places all chemicals, reagents, cultures, and
glassware.
17. Do not pour fluids that are biohazardous down the sink.
Practical Manual of Biochemistry
2
18. It is necessary to wash the glassware with soap and water, then rinse it with
distilled water.
19. Flame transfer loops, wires, or needles for transferring biological material
before and immediately after use.
20. Do not walk around the laboratory with infectious matter containing
transfer loops, wires, needles, or pipettes.
21. Around Bunsen burners, be careful. It is not always possible to see flames.
22. Turn off the incinerators before the laboratory leaves.
23. Report any broken equipment, report any broken glass, in particular those
containing infectious materials immediately.
24. Contact your course instructor or TA immediately if you are injured in the
laboratory.
25. In the event of further treatment being required, spills, cuts and other
accidents should be reported to the instructor or TA.
26. Familiarize yourself with safety equipment and emergency escape routes in
the laboratory.
27. Before putting it away, always wipe and clean your microscope's lenses. To
this end, use the relevant tissue paper and cleaning solution.
28. With all biological fluids, apply appropriate universal precautions.
29. Without the written permission of the course instructor or TA, do not
remove any materials from the laboratory.
Practical Manual of Biochemistry
3
2. BUFFER SOLUTION PREPARATION AND PH
MEASUREMENT
Principal
A buffer's main purpose is to control the solution's pH. Buffers
can also play secondary roles in a system, such as controlling ionic strength
or species solving, perhaps even affecting the structure or activity of protein
or nucleic acid. Nucleic acids, nucleic acid-protein complexes, proteins, and
biochemical reactions are stabilized by buffers (whose products might be
used in subsequent biochemical reactions). Complex buffer systems in
electrophoretic systems are used to control the pH and to establish the pH
gradient. Weak acids and bases are made up of buffer solutions that make
them comparatively resistant to pH change. Theoretically, buffers offer a
ready source of both acid and base to either supply additional H+ if the
process consumes H+ or if a reaction produces acid, combine it with excess
H+.
Reagents:
a. Acetic acid 0.2 M: glacial acetic acid 1.5 ml is made up to 100 ml by using
distilled water.
b. Citric acid: citric acid 2.10 gm in 100 ml distilled water.
c. Dibasic sodium phosphate: 5.3 gm of disodium hydrogen phosphate in
100 ml distilled water.
e. Monobasic sodium phosphate: 2.78 gm sodium dihydrogen phosphate in
100 ml distilled water.
f. Sodium acetate solution: 0.64 gm of sodium acetate in 100 ml distilled
water.
g. Sodium bicarbonate solution: sodium bicarbonate 1.68 gm in 100 ml
distilled water.
h. Sodium carbonate solution 0.2 M:2.12 gm anhydrous sodium carbonate
in 100 ml distilled water.
i. Sodium citrate solution 0.1 M: sodium citrate 2.94 gm in 100 ml distilled
water.
Procedure:
a. Acetic acid-sodium acetate buffer
Take a 100 ml flask and use a pipette to add 36.2 ml of sodium
acetate solution, and then add 14.8 ml of glacial acetic acid to it. Using
Practical Manual of Biochemistry
4
distilled water to produce a total volume of 100 ml. The resulting acetic
acid-sodium acetate buffer is 0.2 M. With the help of a pH meter, the pH is
measured. With distilled water, the electrode is washed, and then dipped in
the prepared buffer solution. The resultant pH is 4.6.
b. Barbitone buffer
In distilled water, mix 2.85 gm of diethyl barbituric acid and 14.2
gm of sodium diethyl barbititrate and then produce 1000 ml of final
volume. That's the buffer of barbitone. With the help of the pH meter, the
pH is measured, and the final pH comes out as 6.8.
c. Citrate buffer
Using distilled water, mix 46.5 ml of citric acid with 3.5 ml of
sodium citrate solution and add 100 ml to the final volume. This is a citrate
buffer of 0.1 M. With the help of a pH meter, the pH of this buffer is
measured and the pH is 2.5.
d. Carbonate-Bicarbonate buffer
Put 27.5 ml of sodium carbonate solution in a flask and add 22.5
ml of sodium bicarbonate solution to it. Then make 100 ml of the total
volume with the aid of distilled water. This is a buffer of 0.2 M carbonate-
bicarbonate. The pH meter is standardized, and the pH of the buffer
solution that is prepared is measured. The pH would be 10.2.
e. Phosphate buffer
Dihydrogen sodium phosphate (39 ml) is mixed with disodium
hydrogen phosphate (61 ml), and 200 ml of distilled water is added to the
final volume. This solution results in the phosphate buffer being 0.2 M.
Using a pH meter, the pH of the phosphate buffer is measured and 6.8 is
obtained.
Preparation of solutions (Molar, Normal) and Dilution
a. Solution Preparation
A solution is a homogeneous mixture produced in a solvent by
dissolving one or more solutes. The smaller chemical, the solute, is soluble
in the solvent (the chemical present in a larger amount). As standard (stock)
Practical Manual of Biochemistry
5
solutions, solutions with precisely known concentrations can be referred to.
These solutions are purchased directly from the manufacturer or formed by
dissolving the desired amount of solute into a specific volume volumetric
flask. Stock solutions are often diluted into lower concentration solutions
for experimental use in the laboratory.
b. Preparing a Standard Solution from a Solid
By two similar methods, a solution of known concentration can be
prepared from solids. Although there are inherent errors with each of the
methods, either for making solutions in the General Chemistry Laboratory
will be sufficient with careful technique.
In the first method, the solid solvent is weighed on paper or in a
small container and then transferred directly to a volumetric flask
(commonly called a "vol flask"). In transferring the solid into the slim neck
of the vol flask, a funnel could be helpful. In the vol flask, a small amount
of solvent is then added and the contents are gently swirled until the
substance is completely dissolved.
More solvent is added until the liquid meniscus reaches the
calibration mark (a process called diluting to volume) on the neck of the
volume flask. Until the contents are mixed and completely dissolved, the
vol flask is then capped and reversed several times. The drawback of this
method is that the original container, weighing paper, or funnel can adhere
to some of the weighed solid. Also, when it is transferred into the slim neck
of the vol flask, solid can be spilled.
In the second method, in a small beaker, the solid is weighed out
first. A small amount of solvent is added to the beaker and until the solid is
dissolved, the solution is stirred. The solution is then moved to the flask of
vol. Again a funnel may need to be inserted into the flask's slender neck.
The beaker, stirring rod, and funnel must be carefully rinsed before
adding additional solvent to the flask, and the washes added to the vol flask
to ensure that all remaining traces of the solution have been transferred.
The vol flask is finally diluted to volume (additional solvent is added to the
Practical Manual of Biochemistry
6
flask until the liquid level reaches the calibration mark). As before the flask
is capped and inverted until the contents are mixed thoroughly. The
downside to this technique is that if not thoroughly washed, some of the
solution may stick to the beaker, stirring rod, or funnel. Also, if they have
not been washed carefully, there is a chance of contamination from the
beaker, rod, or funnel.
c. Molar Solution
It consists of one mole of solvent in a solution equal to one liter.
Molar solution = Molecular weight in the solution in grams / liters.
Example: Sodium chloride molar solution I (NaCl).
Sodium atomic weight = 23
Chloride atomic weight = 35.5
Total molecular weight = 58.5 gram / mol
Now dissolve 58.5 grams of NaCl in distilled water and make the solution
to one liter.
d. Normal Solution
The normal solution is defined as the solution's gram equivalent
weight per liter.
Normal solution = gram equivalent solvent weight/solution liter.
These alternatives are expressed as N.
Gram equivalent weight = weight/valence of molecules
Sodium citrate is widely used for coagulation studies.
For PT and PTT.
The sample can be used for ESR by the Westergren method.
Mechanism of action:
it is used in solution form.
This will chelate calcium. Inactivates Ca++ ions.
This will prevent the rapid deterioration of labile coagulation
factors like factor V and factor VII.
Sodium citrate mechanism as an anticoagulant
Solution preparation and uses:
Trisodium citrate= 3.2 to 3.8 g/dL (3.2% solution).
Mix well Trisodium citrate 3.8 grams in distle water.
Practical Manual of Biochemistry
50
This can be used as 0.109 mg/mL.
In blood, its ratio is 1:9 where 9 parts are blood and 1 part is sodium citrate.
PT and PTT= Blood: Sodium citrate = 9: 1 part (blood 9 parts: sodium
citrate 1 part)
ESR = Blood: Sodium citrate = 4:1 (1.6 mL of blood: o.4 mL Sodium
citrate).
Potassium Oxalate
This may be sodium, potassium, ammonium, or lithium oxalic acid
salt used as an anticoagulant.
This form insoluble complex with calcium ions (precipitate with calcium as
a salt).
Potassium oxalate mechanism as an anticoagulant
This is the most popular oxalate salt used as an anticoagulant in
powder form.
Solution:
Potassium oxalate at a concentration of 1 to 2 mg/mL of blood is
used.
Mix 30 grams/dL in distal watr.
Now add a few drops in the test tube side and dry it in the oven
below 100 °C.
The combination of ammonium/potassium oxalate does not lead
to shrinkage of the RBCs.
While other oxalates cause shrinkage.
Sodium Fluoride
This is a weak anticoagulant but used an antiglycolytic agent to
preserve the glucose.
This inhibits the system involved in glycolysis and preserve the
glucose.
This can be used as a dry additive.
Mechanism of action: It acts in two ways:
As an anticoagulant by binding the calcium.
As an enzyme inhibitor which prevents the glycolytic enzyme to destroy the
glucose.
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51
Sodium fluoride mechanism as an anticoagulant
Solution:
This is effective at a concentration of 2 mg/mL of blood along
with another anticoagulant like potassium oxalate.
When used alone then more concentration than 2 mg/mL is
needed.
This can be used in combination with oxalate as a fluoride-oxalate
mixture.
Most specimens are preserved at 25 °C for 24 hours and at 4 °C for
48 hours.
Sodium fluoride is poorly soluble so mix blood thoroughly before
effective anti-glycolysis occurs. This is mostly used for glucose estimation.
Sodium Iodoacetate
This is also an antiglycolytic agent at a concentration of 2 g/L.
This may be substituted for sodium fluoride.
This has no effect on urease.
Drawback:
It inhibits creatine kinase.
Adverse effects of the additives:
The additive may contain the substance to be tested like Na+oxalate for the
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52
estimation of Na+.
The additive may remove the component to be tested like in oxalate,
removes the calcium.
The additive may affect enzymes like Na+flouride. This may destroy many
enzymes.
A small amount of the anticoagulant gives rise to microclots and
this will interfere with cell count.
The additive may distort the cells like oxalate will change the cell
morphology like RBCs and these will become crenated. While WBCs show
vacuoles. Lymphocytes and monocytes will have distorted shapes.
If the excess quantity is used that will dilute the substance to be tested.
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53
12. TYPE OF COLLECTION PROCEDURES
Capillary blood (skin puncture)
This is good for a small quantity of blood. Warm the finger taking
the blood sample.
In the newborn, under 3 months the heel is the best site to get a
small quantity of blood.
The depth should not be >2.4 mm on the heel.
Avoid the central portion and back of the heel.
Venous blood (venipuncture):
For larger quantities, venous blood will be taken.
The blood sample is taken from the forearm, wrist, or ankle veins.
A forearm site is preferred. Blood is taken directly from the vein, called
phlebotomy.
The median cubital vein is usually preferred.
Mostly venous blood is drawn in the fasting state.
Blood collected after the meal is called a postprandial sample.
There are biological variables in the blood collection like:
Patient lying in the bed or standing up.
After the exercise.
Diurnal variations.
Recent food intake.
Recent intake of Tea/coffee (caffeine), smoking (nicotine), alcohol
ingestion, and administration of the drugs.
Various Blood Samples:
Whole Blood
This blood sample obtained in the test tube containing an
anticoagulant.
This sample will contain cells (white blood cells, platelets, RBCs, proteins)
and plasma.
Plasma
This is a pale yellow liquid which contains RBCs, white cells, and
platelets. Plasma forms with the help of anticoagulants which will prevent
the clotting.
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54
There is the presence of fibrinogen in the plasma.
Serum
This is a clear fluid that is separated from the clotted blood. There
are no RBCs, white cells, or platelets. There is no need for anticoagulants.
Clotted blood is kept at 37 C for at least 20 minutes and then
centrifuged.
The upper portion is called serum.
There is no fibrinogen. serum and plasma difference
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55
13. PROXIMATE ANALYSIS
Moisture
One gram sample of samples were taken in a Petri dish. The weight
of petridish with sample was taken. Then it was placed in the oven at 60ºC
for 12 hours or until it is dried. The dried samples were transferred to
desiccators for minutes and weighted. The samples were again kept in oven
for one to two hours until constant weight (W2) was obtained. The loss in
weight was recorded as moisture.
W1-W2
Moisture (%) =---------------- X 100
W3
Where,
W1 = weight of Petri dish + sample before drying;
W2 = weight of Petri dish + sample after drying;
W3 = weight of the sample.
Total ash
The total ash was determined by burning 2g of feed in a pre-
weighed China dish and then samples were placed in a muffle furnace for
ignition at 550 – 600°C till residue was obtained after 4 – 5 hours. Then the
sample residue were placed in desiccators to cool and weight was recorded.
Percentage of ash was obtained by using the following formula:
Wt. of ash
Total ash (%) = ---------------------------× 100
Wt. of feed
Dry matter
Dry matter content was determined by difference between fresh
weight of sample and moisture content. The crude protein, crude lipid,
crude fibre, nitrogen free extract (NFE), ash contents were calculated on %
dry matter basis. Dry matter percentage was calculated by the following.
Dry matter (%) = 100 - moisture (%)
Nitrogen free Extract
The nitrogen free extract
NFE = 100-(% crude protein + % of crude lipid + % of moisture
+ % ash).
Practical Manual of Biochemistry
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14. ESTIMATION OF REDUCING SUGAR BY
BENEDICT’S METHOD
Principle
Benedict’s quantitative reagent is a modification of qualitative. It
contains copper sulphate, sodium acetate and sodium corbonate. It also
contains potassium thio cyanate and small amount of potassium
ferricyanide. The inclusion of acetate prevents the precipitation of copper
carbonate by chelating Cu3+ion. The thiocyanate causes the precipitation of
white cuprous thio cyanate rather than red cupric oxide. On reduction of
Cu3+ ions which enables the end point of the titration ie., the transition
from blue to white to be readily observable. Methylene blue will be used as
an additional indicator. The small amount of potassium ferricyanide
prevents the re-oxidation of copper. A non-stoicheometric reaction is on
which does not follow a defined pathway and cannot be described by an
equation either quantitatively or qualitatively. The reduction of Cu3+ ions by
sugar is a non-stoicheometric equation and is only constant over a small
range of sugar concentration. To obtain accurate results the volume of
sugar added must be with in 6- 12 ml for 10 ml of benedict’s reagent. If the
preliminary titre value Falls outside this range the sugar solution must be
titrations are repeated.
Reagents Required:
Standard Glucose Solution: 200 mg of glucose was weighed accurately and made upto 100 ml
with distilled water (concentration: 2 mg / ml)
Benedict’s Quantitative Reagent
100 ml of solution acetate, 37.5 g of sodium carbonate and 62.5 g of
potassium thiocyanate were dissolved in 300 ml of distilled water by
warming gently and filtered. 9 g of copper sulphate is dissolved in 50 ml of
water, added with continuous stirring. 2.5 ml of potassium ferricyanide is
added and the volume is made upto 500 ml with water.
Anhydrous Sodium Carbonate Procedure
100 ml of benedict’s reagent was pipetted out into a clean conical
flask. About 600 mg of anhydrous sodium carbonate was added to
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57
provide the required allcaling with A few porcelain bits and heated to
boiling over a moderate flame.
The standard glucose solution is taken in the burette when the
benedict’s solution Boils, glucose solution is added drop by drop (one
drop per second) till the last trace of blue colour disappears. The volume
of glucose rundown is noted and the titrations are repeated for concordant
value.
The given unknown sugar solution was made upto 100 ml in a
standard flask with distilled water. Then the burette was filled with
unknown sugar solution and the benedict’s reagent was titrated as before.
The volume of sugar solution rundown was noted and titrations are
repeated for concordant values.
Estimation Of Reducing Sugar By Benedict’s Method
Titration 1
Standardisation Of Benedict’s Reagent Benedict’s Reagent Vs
Standard Glucose Solution
S.No Volume of
Benedict’s
reagents(ml)
Burette Readings Volume of
standard
glucose(ml)
Indicator
Intial
ml
Final
ml
Self
TITRATION 2:
Estimation Of Glucose Standardised Benedict’s Reagent Vs Unknown
Glucose
S.No Volume of
Benedict’s
reagents(ml)
Burette Readings Volume of
unknown
glucose(ml)
Indicator
Intial
ml
Final
ml
Self
Practical Manual of Biochemistry
58
Calculation:
The standard glucose solution 2 mg / ml 5 ml of Benedict’s solution react with…………..ml of the standard glucose solution. ……….ml of standard glucose solution which contains x 2 = mg 5 ml of Benedict’s solution reacts with…….. mg of unknown glucose 100 ml of unknown glucose contains is
100 x……..
Result:
The amount of glucose present in 100 ml of given unknown
solution is………..
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59
15. ESTIMATION OF PROTEIN BY LOWREY’S METHOD
Principle: Protein in the given solution when treated with alkaline copper
sulphate and Folin’s phenol reagent produces a blue colored complex. The
intensity of the colour is directly proportional to the concentration of
protein present in the given sample solution.
Reagents Required
Stock Solution:
Bovine serum albumin of 100mg is weighed accurately and
dissolved in 100ml of distilled water in a standard flask.
(Concentration:1mg/ml)
Working Standard:
The stock solution of 10ml is diluted to 100ml with distilled water
in a standard flask. (Concentration:100mg.ml)
Folin;s Phenol Reagent:
Folin’s phenol reagent is mixed with distilled water in a the ratio
1:2
Alkaline CuS04 Reagent: Solution A:
Sodium carbonate of 2% in 0.1N sodium hydroxide.
Solution B: Sodium Potassium tartrate of 1%
Solution C: Copper sulphate of 0.5%
Solutions A, B, C are mixed in the proportion of 50:1:0.5
Unknown Preparation:
The given protein is made upto 100ml with distilled water.
Procedure:
Working standard of 0.2 to 1.0ml is pipetted out into clean test
tubes labelled as S1, to S5 . Test solution of 0.2 and 0.4 ml is taken in test
tubes labelled as T1 and T2. The volume is made upto 1.0ml with distilled
water. Distilled water of 1.0ml serves as blank. To all the test tubes 4.5 ml
of alkaline copper sulphate reagent is added and it is incubated at room
temperature for 10 minutes. To all the test tubes 0.5ml of Folin’s Phenol
reagent is added. The contents are mixed well and the blue colour
developed is read at 640nm after 15 minutes. From the standard graph the
amount of protein in the given unknown solution is calculated.
Practical Manual of Biochemistry
60
Estimation of Protein by Lowrey’s Method
S.No Reagents B S1 S2 S3 S4 S5 T1 T2
1 Volume of working standard (ml)
- 0.2 0.4 0.6 0.8 1.0 - -
2 Concentration of working standard (mg)
- 20 40 60 80 100 - -
3 Volume of unknown solution (ml)
- - - - - - 0.2 0.4
4 Volume of distilled water (ml)
1.0 0.8 0.6 0.4 0.2 - 0.8 0.6
5 Volume of alkaline copper reagent (ml)
4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5
6 Volume of Folin’s phenol reagent
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
The contents are mixed well and kept at room temperature for 10 minutes. The blue colour developed is read at 640nm
7 Optical density 640nm
Calculation:
………of unknown solution corresponds to……..xOD
OD corresponds of ……….. mg of protein
i.e. 0.2ml of unknown solution contains ……….of protein 100ml of
unknown solution contains
100 x ----
Result:
The amount of protein present in the given solution is…………
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16. ESTIMATION OF CHOLESTEROL BY ZAK’S METHOD
To estimate the amount of cholesterol in an unknown food sample.
Principle: Cholesterol in glacial acetic acid gives a red colour with ferric
chloride and apolar sulphuric acid. This reaction has been employed by
ZAK’S to estimate the cholesterol in an unknown food sample.
Reagents Required:
Stock Standard Solution :
About 100 mg of cholesterol was dissolved and made up to 100 ml
with glacial acetic acid (concentration 1 mg / ml).
Working Standard :
About 4 ml of stock solution was made up to 100 ml with ferric
chloride acetic acid reagent (concentration in 40 mg / ml).
Ferric chloride of 0.05% in acetic acid.
Apolar sulphuric acid.
Glacial acetic acid.
Preparation of unknown sample:
20 ml of sample and 40 ml of chloroform was added and
centrifuged. The supernatant was used for estimation.
Procedure:
0.5 ml to 2.5 ml of working standard were Pipetted out into a clean
test tubes. About of 0.5 ml and 1 ml of unknown food sample supernatant
was taken in a test tubes. The volume was made upto 5.0 ml with ferric
chloride and 3.0 ml of concentrated sulphuric acid were added. The test
tubes were kept at room temperature for 15 minutes. The pinkish red
colour formed was measured at 540 nm. Standard graph was drawn for the
values obtained. From the standard graph the amount of cholesterol
present in the food sample can be calculated.
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Estimation of Cholesterol by Zak’s Method
S.N o
Reagents B S1 S2 S3 S4 S5 T1 T2
1. Volume of standard chloresterol (ml)
_ 0.5 1.0 1.5 2.0 2.5 _ _
2. Concentration of cholesterol (mg)
_ 20 40 60 80 100 _ _
3. Volume of sample supernatant (ml)
_ _ _ _ _ _ 1 1
4. Volume of 0.05% ferric chloride acetic acid reagent (ml)
5 4.5 4 3.5 3 2.5 _ _
5. Volume of cone sulphuric acid (ml)
3 3 3 3 3 3 3 3
Incubated the tubes for 15 minutes at room temperature
6. O.D at 540 nm Calculations
0.5 ml of standard corresponds to O.D 0.03 O.D corresponds to……..mg.
0.5 ml of unknown corresponds 100 ml of unknown corresponds to………..
100x………..
Result:
The amount of cholesterol present in unknown food sample was
found to be……………….
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17. ESTIMATION OF DNA BY DIPHENYLAMINE METHOD
Principle:
The deoxyribose of DNA in the presence of acid forms hydroxyl
levolinic aldehyde which reacts with diphenylamine to give a blue colour.
But only the deoxy ribose of purine nucleotide react.
Reagents required:
Stock standard solution: Weighed 100 mg of DNA and dissolved in 100 ml
of distilled water.
Working standard solution: 10 ml of stock was diluted to 100 ml using
distilled water.
Diphenyl amine reagent:
It should be prepared freshly by dissolving 1 gm of diphenyl amine
in 100 ml of glacial acetic acid and by adding 2.5 ml of concentrated
sulphuric acid.
Procedure:
Pipetted out 0.2 ml – 1.0 ml of DNA solution into a series of test
tubes and made up the volume to 3.0 ml with distilled water. 0.2 ml and 0.4
ml of unknown is taken and made upto 3.0 ml with water. Added 5.0 ml of
disphenylamine reagent, mixed well and is heated in a boiling water bath for
10 minutes. Cooled and the colour developed is read at 595 nm.
Calculations
0.2 ml of unknown corresponds to 0.02 O.D
---O.D corresponds to ----
0.2 ml of unknown corresponds to ----
100 ml of unknown corresponds to = 100x………..
Results:
The amount of DNA present in 100 ml of given unknown solution
is……………
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Estimation of Dna by Diphenylamine Method
S.No Contents B S1 S2 S3 S4 S5 U1 U2
1. Volume of working standard in (m1)
- 0.2 0.4 0.6 0.8 1.0 - -
2. Concentration in g - 20 40 60 80 100 - -
3. Volume of unknown
in (m1)
- - - - - - 0.2 0.4
4. Volume of distilled
water in (ml)
3 2.8 2.6 2.4 2.2 2.0 2.8 2.6
5. Volume of diphenyl amine reagent in (ml)
5 5 5 5 5 5 5 5
Heated in a boiling water bath for 10 minutes
6. Optical density at
595 mm
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18. SEPARATION OF AMINO ACIDS BY THIN
LAYER CHROMATOGRAPHY
Principle:
Chromatography is a method by which a mixture of substances in
smaller quantities can be separated both qualitatively and quantitatively. In
chromatography there are twophases-the stationary phase and other mobile
phase. When the mobile phase moves along stationary phase, separation of
substances takes place. In thin layer chromatography, the thin layer of gel
functions as an inert supporting material. When the mobile phase moves
along the gel solvent, as the partition coefficient differ for different sugars,
the rate of flow differs and therefore separation occurs.
Materials Required:
Silica gel G 2.Microscopic slides 3.n-butanol
Acetic acid
Spraying reagent (0.3% solution of Ninhydrin in butanol containing
3 ml acetic acid)
Amino acids
Procedure:
A slurry of silicagel G is prepared in 0.02M sodium acetate buffer.
Taken the microscopic slides and keeping them flat, pipetted out about 1-2
ml of the slurry into them. By tilting the slides spread the slurry evenly on
the surface. Lining the edges with Vaseline will be of help. Allowed the
slides to dry completely leaving them flat. 5l samples of amino acid (or
mixture) are spotted and the slide is then dipped in a trough containing n-
butanol-acetic acid-water in the ration 8:2:2. The slide must be handled with
care not to break the surface. After development, that is, when the solvent
has reached the top, the slide is dried and sprayed with the developing
reagent. The slide is then heated in an oven at 1100 C for 10 minutes and
Rf values of the spots are measured.
Distance moved by solute
Rf =-------------------------------------------
Distance moved by solvent
Result:
The given sample Rf value is ……………………
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19. SEPARATION OF SUGARS BY PAPER
CHROMATOGRAPHY
Principle:
Chromatography is a method by which a mixture of substances in
smaller quantities can be separated both qualitatively and quantitatively. In
chromatography there are two pahses the stationary phase and other mobile
phase. When the mobile phase moves along stationary phase, separation of
substances takes place. In paper chromatography the paper functions as an
inert supporting material. When the mobile phase moves along the paper
solvent, As the partition coefficient differ for different sugars the rate of
Stock cholesterol solution (2 mg/mL in chloroform)
Working cholesterol solution (dilute the previous solution in 1:5
ratios in chloroform to give a strength of 0.4 mg/mL).
Protocol
Place 10 mL of the alcohol–acetone solvent in a centrifuge tube,
and add 0.2 mL of serum or blood.
Immerse the tube in a boiling water bath with shaking until the
solvent begins to boil. Remove the tube and continue shaking the mixture
for a further 5 min. Cool to room temperature and centrifuge.
Decant the supernatant fluid into a test tube and evaporate to
dryness on a boiling water bath.
Cool and dissolve the residue in 2 mL of chloroform. At the same
time, set up a series of standard tubes containing cholesterol and a blank
with 2 mL of chloroform.
Add 2 mL of acetic anhydride–sulfuric acid mixture to all tubes and
thoroughly mix. Leave the tubes in the dark at room temperature and read
the extinction at 680 nm.
Results
The normal serum cholesterol lies within the range of 100–250
mg/100 mL.
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35. ISOLATION OF RNA FROM YEAST
Principle
Total yeast RNA is obtained by extracting a whole-cell homogenate
with phenol. The concentrated solution of phenol disrupts hydrogen
bonding in the macromolecules, causing denaturation of the protein. The
turbid suspension is centrifuged, and two phases appear: the lower phenol
phase contains DNA, and the upper aqueous phase contains carbohydrate
and RNA. Denatured protein, which is present in both phases, is removed
by centrifugation. The RNA is then precipitated with alcohol. The product
obtained is free of DNA but usually contaminated with polysaccharide.
Further purification can be made by treating the preparation with amylase.
Materials
Dried yeast, phenol solution (90%),
Potassium acetate (20%, pH 5),
Absolute ethanol, diethyl ether
Protocol
Suspend 30 g of dried yeast in 120 mL of water previously heated
to 37°C. Leave for 15 min at this temperature and add 160 mL of
concentrated phenol solution.
Stir the suspension mechanically for 30 min at room temperature,
and then centrifuge at 3000g for 15 min in the cold to break the emulsion.
Carefully remove the upper aqueous layer with a Pasteur pipette, and
centrifuge at 10,000g for 5 min in a refrigerated centrifuge to sediment
denatured protein.
Add potassium acetate to the supernatant to a final concentration
of 20 g/L, and precipitate the RNA by adding two volumes of ethanol.
Cool the solution in ice and leave to stand for 1 h.
Collect the precipitate by centrifuging at 2000g for 5 min in the
cold. Wash the RNA with ethanol–water (3:1), ethanol, and, finally, ether;
air-dry and weigh. (Note: Yeast contains about 4% RNA by dry weight.)
Compare with a commercial preparation by measuring the pentose,
phosphorus, and DNA content and by determining the absorption
spectrum. Keep your preparation for use in later experiments.
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36. ESTIMATION OF CHLOROPHYLL
CONCENTRATION IN THE CHLOROPLAST
SUSPENSION
Principle
Pigments absorb light to different extents depending on the
exposure of light of varying wavelength of the light spectrum. The ideal
wavelength to select is the peak of the pigment’s absorption spectrum for
quantification of a pigment. For example, for chlorophyll a, 663 nm is
generally used, but for chlorophyll b, 645 nm is a more appropriate
wavelength to choose. The greater the concentration of a pigment in solu-
tion, the larger the proportion of light absorbed by the sample at that
wavelength.
Procedure
Take 1 mL aliquot of the chloroplast suspension into a 10 mL
graduated cylinder, and dilute to 10 mL with 80% acetone. Cover the
cylinder with parafilm and mix by inverting.
Prepare the spectrophotometer to read the absorbance of the diluted
chlorophyll extract.
Adjust the wavelength to read 652 nm (why is this wavelength
chosen?). Without a cuvette in the machine, adjust to 0% transmittance
(left-hand knob).
Blank the spectrophotometer with the reagent blank (80% acetone) to
read 0 absorbance (right-hand knob). Transfer some of diluted chlorophyll
extract to a cuvette and read the absorbance. Record the absorbance value.
Chlorophyll absorbance (A652) = OD
Calculate the chlorophyll content in the diluted sample using the
following equation. Record the chlorophyll concentration of the
diluted sample.
A = ECd where A is the observed absorbance, E is a proportionality
constant (extinction coefficient) (=36 mL/cm), C is the chlorophyll
concentration (mg/mL), and d is the distance of the light path (=1
cm).
Calculate the chlorophyll concentration in the original chloroplast
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94
suspension (undiluted) by adjusting for the dilution factor. In order
to determine the concentration of chlorophyll in the original
suspension, you must multiply the chlorophyll concentration in the
diluted sample by the dilution factor.
Knowing the chlorophyll content of the undiluted chloroplasts, prepare
10 mL of chloroplast suspension containing approximately 0.02
mg/mL chlorophyll by diluting an appropriate aliquot of original
chloroplast suspension with cold 0.5 M sucrose. Keep this on ice.
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37. DETERMINATION OF TOTAL ERYTHROCYTE
COUNT
For RBC count, a method devised by Yokayama (1974) and later
modified by Christensen et al., (1978) was followed. Hayems diluting fluid,
which had the following composition, was used for RBC count.
Chemicals:
Mercuric chloride : 0.5gm
Sodium chloride : 1.0gm
Sodium sulphate : 5.0 gm
Distilled water : 200ml
Procedure
An improved Neubauer,s counting chamber was used for counting
RBC (Baker and Silverton, 1982).
Using RBC pipette, the blood was drawn upto 0.5 mark and the
diluting fluid to the mark 101.
Although fluid is drawn to the mark 101 but the real dilution is
0.5:100 or 1:200 because the fluid in the capillary tube is discarded before
the count.
Calculations
The number of RBC’s per sq mm was calculated as follows:
Area of a small square : 1/400 sq mm
Depth of the counting chamber : 1/10mm
The volume of Small Squareis : 1/4000cumm
The dilution of the blood is : 1/200
Total RBC = cu mm
N = No of cells in 80 small squares
Result
The total RBC present in the given sample is ……………….
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38. DETERMINATION OF TOTAL LEUCOCYTE COUNT
A white cell count (TLC) estimates the total number of white cells
in a cubic millimetre of blood. WBC diluting fluid or Turk fluid contains a
weak acid to lyse the red blood cells and Gentian violet stain for staining
the nucleus of white blood cells.
Chemicals:
The Turks fluid with following composition was used for TLC:
Glacial acetic acid :1.5ml
1% aqueous solution of Gentian violet :1 ml
Distilled water :100ml
Procedure
Neubauer,s hemocytometer (Baker and Silverton, 1982) was used
for counting of leucocytes.
Using white cell pipette, the blood was drawn upto 0.5 mark and
the diluting fluid to 11 mark, thus the dilution was 1:20.
Calculations
The number of RBC’s per sq mm was calculated as follows:
Area of a small square : 4 sq mm
Depth of the counting chamber : 1/10mm
The dilution of the blood is : 1/20
N x 20/10
Total WBC = ----------------- cu mm
4
Result
The total WBC present in the given sample is ……………….
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39. DETERMINATION OF PACKED CELL VOLUME
(PCV) OR HAEMATOCRIT
PCV was determined by micro haematocrit method of Schalm et
al., (1975).
Chemicals:
EDTA blood
PCV tube
Procedure
The heparinised blood was filled upto the mark 100 of the
haematocrit tube with the help of Pasteur pipette and centrifuged at 3000
rpm for 30minutes.
The relative volume of the height of the RBC’s packed at the
bottom of the haematocrit tube was recorded as (PCV) in terms of
percentage of total blood column taken in the haematocrit tube.
Result
The PCV or Haematocrit present in the given sample is
……………….
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40. DETERMINATION OF MEAN CORPUSCULAR
VOLUME (MCV)
MCV indicates the average size of the RBC in a given sample of
blood.
MCV was calculated by the following formula and represented in
cubic microns.
MCV (fl) = PCV×10
RBC Count
Result
The MVC present in the given sample is ……………….
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99
41. DETERMINATION OF MEAN CORPUSCULAR
HEMOGLOBIN (MCH)
MCH represents the average weight of the hemoglobin contained
in each RBC in a given volume of the blood.
MCH is influenced by the size of the cell and concentration of the
hemoglobin.
MCH was calculated by the following formula and expressed in
pictograms (pg).
MCH (pg) = Hemoglobin(g / dL)×10
RBC Count
Result
The MCH present in the given sample is ……………….
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42. MEAN CORPUSCULAR HEMOGLOBIN
CONCENTRATION (MCHC)
MCHC reflects the average concentration of the hemoglobin in the
RBC in a given volume of the blood.
MCHC was obtained by the following formula and expressed in
terms of percentage.
MCHC (%) = Hemoglobin(g / dL)
×100PCV
Result
The MCHC present in the given sample is ……………….
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43. DIFFERENTIAL LEUKOCYTE COUNT
Differential Leucocyte Count in blood was determined by the
method Ghai, (1993). A dried blood film stained with Leishman’s stain, was
examined under oil a immersion objective and the different type of white
blood cells were identified. The percentage of distribution these cells was
then determined.
Reagents
Leishman’s stain (Eosin and methylene blue dyes dissolved in
acetone-free absolute methyl alcohol).
Procedure
A blood film was prepared, dried and placed on the staining rack
and was covered with Leishman’s stain, allowed to stand for 2 minutes.
Then equal volume of distilled water was added and mixed by the
slide first one way and then other.
Allowed to stand it for 6 minutes. Drained off diluted stain in a
stream of distilled water from a wash bottle for about 20 seconds and
allowed the slide to remain on the staining rack for 1-2 minutes with the last
wash covering it.
Then kept the slide against a support in an inclined position,
stained smear facing down and allowed it to dry.
Then the stained slides were studied under low and high power
objectives for differential leucocyte count by placing two drops of glycerol
on the stained smear and using oil-immersion lens.
Calculations
Differential leucocyte was expressed as percentage.
Number of type cells
DLC (%) = -------------------------------------------------- x 100
Total number of leukocytes
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44. DETERMINATION OF SERUM AMYLASE Principle:
Serum is incubated with starch substrate. The amylase in the serum
hydrolyses the starch to simpler units with a resulting increase in reducing
groups. In the method presented here iodine is added which reacts with the
starch molecules not hydrolysed by the amylase. The iodine-starch complex
is blue in colour and is measured in the spectrophotometer. The degree of
loss in colour is proportional to the amount of starch hydrolysed and hence
to the activity of the amylase in the serum. A substrate control is carried
though the procedure to give a reference value for the amount of starch
substrate present before hydrolysis.
Procedure 1. Pipette 5 ml of substrate into two 50 ml volumetric flasks. 2. Place the 'test' flask into a 34oC water bath for 5 minute to warm the contents. 3. Using a pipette that deviloers between two marks add 0.1 ml of serum to the 'test' flask and mix. Do not use blow out pipettes as the smallest amount of saliva can give a large error. 4. Time the addition of serum using a stop watch. 5. After exactly 7.5 minutes add 5 ml or working iodine solution, mix and immediately remove from the water bath. 6. Similarly add 5 ml of the working iodine solution to the flask containing the 'substrate control', which has not been incubated. 7. Dilute the contents of both flasks to the 50 ml mark with distilled water and mix the flasks well. 8. Read the absorbance of both against water using the large (19 mm) cuvettes at 660 nm. Calculation Absorbance of substrate control – absorbance of test x 800 Absorbance of control units of amylase activity per 100 ml of serum.
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45. ESTIMATION OF SERUM URIC ACID
Principle:
Phosphotungstic acid in alkaline medium oxidizes uric acid
to allantion and itself gets reduced to tungsten blue which is
estimated colorimetrically at 700mm.
Reagents :
Sodium tungstate 10%.
2/3 N Sulphuric acid.
Tungstic acid: Add 50ml of 10% sodium tungstate 50ml 2/3 N H2SO4 and
a drop of phosphoric acid with mixing to 800ml water. Discard when
cloudy. Store in brown bottle.
Phosphotungstic acid: Stock-Dissolve 50g sodium tungstate in about 400ml
of water. Add 40ml 85% phosphoric acid and reflux gently for 2 hours,
cool, make volume to 500m. store in brown bottle. Dilute 1 to 1 for use.
Na3CO3 10%.
Standard uric acid solution stock-100mg%.
Working uric acid solution-1mg%.
Procedure :
In a centrifuge tube pipette 0.6ml serum and add 5.4ml. tungstic
acid while shaking. Centrifuge and process as follows.
B T S S2 S3
1. Standard uric acid - - 1.0 2.0 3.0
(1mg%) ml.
2. Supernatant (ml) - 3.0 - - -
3. D. Water (ml) 3.0 - 2.0 1.0 -
4. Na3CO3 (ml) 0.6 0.6 0.6 0.6 0.6
5. Phosphotungstate (ml) 0.6 0.6 0.6 0.6 0.6
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Mix well stand at room temperature for 30 min. Read
absorbance at 700 nm or using a red filter plot a standard curve
between concentration of standard and absorbance and calculate the
uric acid conc, in test.
O.D. Test Conc. of Std. Serum Uric acid =-------------------x--------------------x100 O.D. Std. Vol. of Serum
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46. PHYTOCHEMICAL ANALYSIS
The chemicals produced by plants are referred to as plant
chemicals. These are generated by the primary and secondary metabolism
of the plant. These phytochemicals are essential for other plants, animals,
insects and microbial pests and pathogens to prosper or thwart them. Plants
are also assisted and protected from disease and harm caused by
environmental hazards such as pollution, UV, stress and draught. They are
used as conventional medicine and as ancient poisons.
Phytochemicals are not the essential nutrients they are, rather than
the essential nutrients because there is no evidence that they are not yet
established to cause any possible health effects in humans. They are known
to have a role in the protection of human health. More than 4,000
phytochemicals are classified by protective function, physical characteristics
and chemical characteristics and have been catalogued. Phytochemicals are
usually classified into the following types; carotenoids and polyphenols,
including phenolic acids, stilbenes/lignans, are included. In addition,
flavones, anthocyanins, isoflavones and flavanols are further categorized
into classifications such as flavonoids.
Steps in Preparation of Plant Sample
Drying
The plant materials are dried to remove the water content and thus
to store them after the water is removed. As soon as the plants are
collected, this process should be done immediately so that spoilage is
prevented. In drying the plants, there are two techniques,
Natural process
This process include sun-drying. In this the plants are kept in the
shades and are air dried in sheds. This process takes few weeks for
complete drying of the moisture. This time depends on the temperature and
humidity.
Artificial drying
Using the help of artificial driers, artificial drying is done. The time
consumed is reduced to a few hours or minutes by this process. Warm-air
drying is the common method used for the drying of medicinal plants. The
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106
hot air furnace on which hot air is blown is used to do this. For drying
succulent parts of plants and fragile flowers, this method is applicable. To
avoid disintegration of the thermolabile compounds, the drying must be
done at a lower temperature.
Grinding of plant materials
The plant samples are to be powdered for further analysis
following complete drying of moisture. There are various forms of
powdering, including the following,
1. Grinding can be done by grinding in an electric grinder or a spice mill or
in a mortar or pestle as well.
2. Due to the increased surface area of the plants, grinding increases the
effectiveness of extraction. The reduction in the area of the surface can lead
to dense packing of the material.
3. It is always ideal to mill the plants into a fine powder, but if they are too
fine, this impacts the flow of the solvent and also generates more heat that
could degrade some thermolabile compounds.
Choice of Solvent
For the determination of biologically active phytochemicals from
plants, the solvent that is used for the extraction process is very important.
These solvents must be less toxic, easy to evaporate in less heat, preserve
and not dissociate the compounds in them. For extraction, the different
solvents commonly used include:
1. Water:
It is a universal solvent; plant extracts are usually extracted with
water for anti-microbial activity. But when compared to water, the organic
solvents give consistent results in anti-microbial activities. No significant
results can be obtained from water soluble compounds in the extract.
2. Alcohol:
Due to the presence of higher amounts of polyphenols, these
alcoholic extracts from plants show more activity than aqueous extracts.
This is due to the higher alcohol degradation of the cell wall and seed,
which releases the polyphenols that will be degraded if aqueous extracts are
extracted. Ethanol, however is microbicidal rather than water. 70 percent
ethanol is used to extract more bioactive compounds than pure ethanol.
Intracellular ingredients from plant materials are also found to be easier to
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107
extract from ethanol. For the extraction of phenolic compounds, polar
solvents like methanol, ethanol and their aqueous mixtures are used. The
addition of water to alcohol will enhance the extraction rate. Because of its
cytotoxic nature, methanol is more polar but is unsuitable for extraction.
3. Acetone:
Acetone dissolves many of the plants' hydrophilic and lipophilic
compounds and is water-mixable. It is low in toxicity and volatile and is
used to extract antimicrobial activity. Tannins and other phenolic
compounds are extracted using acetone. In addition, they are used to
extract saponins.
4. Chloroform:
Terpenoid lactones are obtained from barks by extraction with
chloroform. Tannins and Terpenoids are treated with less polar solvents.
5. Ether: They are used for the extraction of coumarins and fatty acids.
Methods of Extraction:
Homogenization
One of the most commonly used methods for extraction is this
technique. This is done using either the dried or wet method of extraction.
The dried plant samples are finely powdered in this dried extraction method
and added to the solvent blended for a few minutes and kept for about 24
hours in an orbital shaker. The parts of the plants are cut into small pieces
during the wet extraction process, ground in a mortar and pestle and added
to a solvent and shaken for 24 hours in an orbital shaker and then filtered.
The filtrate can be used for the further analysis.
Serial Exhaustive Extraction
It is done to extract a wide range of polarities of compounds with a
variety of solvents from a non-polar solvent such as hexane to more polar
solvent such as methanol. The drawback is that due to the high heat leading
to the degradation, thermolabile compounds can not be extracted.
Soxhlet Extraction
It is used when the compound in the solvent is less soluble and the
impurities in the solvent are soluble. The impurities can be eliminated by
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108
simple filtration if the desired compound is highly soluble in the solvent.
The benefit is that the solvent is recycled in this method and therefore the
solvent is less wasted. Thermolabile compounds cannot be extracted in this
method, similar to the above method.
Maceration In this method,
With frequent agitation, the entire plant or the powder can be kept
in the solvent for a certain period until the soluble compounds are
dissolved. For thermolabile compounds, this method is the most
appropriate method.
Decoction
Heat-stable and water-soluble compounds are extracted in this
process. The plant materials extracted are cooked in the water for about 15
minutes and cooled, filtered and used for further analysis. 4.6. Infusion
By diluting the compounds in the solvents, it is done. It is prepared by
macerating the compounds in cold or boiling water for a brief period.
Digestion
This is a process where the extraction is done as maceration with a
gentle heat applied. It is used when the elevated temperature do not
interfere the solvent efficiency or the compounds.
Percolation
For this process an instrument called percolator is used which is a
narrow, cone shaped Vessel with open ends. The ingredients are moistened
with an appropriate amount of the specified menstrum and allowed to stand
for approximately 4 h in a well closed container, after which the mass is
packed and the top of the percolator is closed. Additional menstrum is
added to form a shallow layer above the mass, and the mixture is allowed to
macerate in the closed percolator for 24 h. The outlet of the percolator then
is opened and the liquid contained therein is allowed to drip slowly.
Additional menstrum is added as required, until the percolate measures
about three quarters of the required volume of the finished product. The
marc is then pressed and the expressed liquid is added to the percolate.
Sufficient menstrum is added to produce the required volume, and the
mixed liquid is clarified by filtration or by standing followed by decanting.
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Sonication
In this method the ultrasound with higher frequencies of 20 kHz –
2000 kHz are used which will disrupt the cells and releases the constituents.
Although the process is useful in some cases, like extraction of rauwolfi a
root, its large-scale application is limited due to the higher costs. One
disadvantage of the procedure is the occasional but known deleterious
effect of ultrasound energy (more than 20 kHz) on the active constituents
of medicinal plants through formation of free radicals and consequently
undesirable changes in the drug molecules.
Qualitative Analysis of Primary Metabolites:
Test for carbohydrates
Benedict’s test:
About 0.5 ml of the filtrate was taken to which 0.5 ml of Benedict’s
reagent is added. This mixture was heated for about 2 minutes in a boiling
water bath. The appearance of red precipitate indicates the presence of
sugars
Molisch’s test:
To about 2ml of the sample, 2 drops of alcoholic solution of α-
napthol was added and to the mixture after being shaken well. Few drops of
conc.H2SO4 were added along the sides of the test tube. A violet ring
indicates the presence of sugars
Test for Starch
To about 5 ml of distilled water, 0.01g of iodine and 0.075 g of
potassium iodide were added and this solution was added to about 2-3 ml
of the extract. Formation of blue colour indicates the presence of starch.
Test for proteins
Biuret test:
2ml of filtrate was taken to which 1 drop of 2% copper sulphate
solution was added; 1ml of 95% ethanol was added. Then it was followed
by excess addition of KOH. The appearance of pink colour indicates the
presence of protein.
2ml of extract was mixed with 2ml of water and about 0.5% of
conc. HNO3 was added. The appearance of yellow colour indicates the
presence of proteins.
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To about 2 ml of the extract, 2ml of miller’s reagent was added
white precipitate which turns red on heating will confirm the presence of
proteins.
Test for amino acids
To 1ml of the extract, few drops of ninhydrin reagent (10mg of
ninhydrin in 200ml of acetone) were added. The appearance of purple
colour indicates the presence of amino acids.
To 2ml of extract few drops of nitic acid were added along the
sides of the tube the appearance of yellow colour indicates the presence of
protein and free amino acids.
Test for fatty acids
1 ml of the extract was mixed with 5 ml of ether. There extracts
were allowed to evaporate on a filter paper and the filter paper was dried.
The appearance of transparency indicates the presence of fatty oils
Miscellaneous compounds
Test of resins
1. Precipitation test: about 0.2 g of extract was extracted with 15ml of 95%
ethanol. The alcoholic extract was then poured into a beaker containing
about 20ml of distilled water.
2. 1ml of extract was taken and to this few ml of acetic anhydride was
added to this 1ml of conc.H2SO4 was added. The appearance of orange to
yellow colour indicates the presence of resins
Test of fixed oils and fats
1. Spot test: small quantity of the extract was taken and pressed between 2
filter papers. The appearance of spots indicates presence of oils
2. Saponification test: To the extract, few drops of 0.5N alcoholic KOH
and few drops of phenolphthalein were added. This mixture was heated for
about 2 hours. The formation of soap or partial neutralization of alkali
indicates the presence of fixed oils or fats
Gums and mucilage
To 1ml of extract, distilled water, 2ml of absolute ethanol was added with
constant stirring white or cloudy precipitate indicates the presence of gums
or mucilage
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Carboxylic acids
1. To 1ml of extract a pinch of sodium bicarbonate is added. The
production of effervescence indicates the presence of carboxylic acids
2. 2ml of alcoholic extract was taken in warm water and filtered. The
filtrate was then tested with litmus paper and methyl orange. The
appearance of blue colour.
Qualitative Analysis of Secondary Metabolites
Test for anthraquinones
To 5ml of extract, few ml of conc.H2SO4 was added and 1ml of
diluted ammonia was added to it. The appearance of rose pink confirms the
presence of anthraquinones
Test for quinones
To 1ml of extract, alcoholic KOH is added the presence of red to
blue colour indicates the presence of quinones
Test for alkaloids
1. Mayer’s test: to a few ml of filtrate, 2 drops Mayer’s reagent was added a
creamy or white precipitate shows a positive result for alkaloids.
2. Wagner’s test (iodine – potassium iodine reagent): To about an ml of
extract few drops of Wagner’s reagent were added. Reddish – brown
precipitate indicates presence of alkaloids.
3. To 5ml of extract 2ml of HCl was added. Then 1 ml of Dragendroff‟s
reagent was added an orange or red precipitate shows a positive result for
alkaloids.
Test for glycosides
1. Borntrager’s test: to 2ml of filtrate, 3ml of chloroform is added and
shaken. The chloroform layer is separated and 10% ammonia solution was
added. The pink colour indicates the presence of glycosides
2. 5ml of extract was hydrolysed with 5ml of conc. HCl boiled for few
hours in a boiling water bath, small amount of alcoholic extract was
dissolved in 2ml of water and 10% of aqueous 10% NaOH was added the
presence of yellow colour was a positive result for the glycosides.
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3. 2ml of extract is mixed with about 0.4 ml of glacial acetic acid containing
traces of ferric chloride and 0.5 of conc. H2SO4 was added the production
of blue colour is positive for glycosides.
Test for cardiac glycosides (Keller-Killani test)
1. 5ml of solvent extract was mixed with 2ml of glacial acetic acid and a
drop of ferric chloride solution was added followed by the addition of 1ml
of conc. H2SO4. A brown ring in the interface indicates the presence of
deoxy sugars of cardenoloides. A violet ring may appear beneath the brown
ring while acetic acid layer a green ring may also form just gradually towards
the layer.
Test for phenol
1. Gelatine test:
To 5ml of extract 2ml of 1% solution of gelatine containing 10% of NaCl is
added. Appearance of white precipitate indicates the presence of phenol
2. Lead acetate test: To 5 ml of extract 3ml of 10%lead acetate solution was
added and mixed gently. The production of bulky white precipitate is
positive for phenols. Test for polyphenols 1. To the 3ml of extracts 10ml of
ethanol was added and were warmed in a water bath for 15 minutes. To this
few drops of ferric cyanide (freshly prepared) was added. The formation of
blue – green colour indicates presence of polyphenols.
2. To 1ml of extract few drops of 5% solution of lead acetate was added.
The appearance of yellow precipitate indicates the positive results for
polyphenols
3. To the 5ml of ethanolic extract 3ml of 0.1% gelatine solution was added.
The formation of precipitate was positive for polyphenols
Test for tannins
1. To 5ml of extract few drops of neutral 5% ferric chloride solution was
added, the production of dark green colour indicates the presence of
tannins
Test for Flavonoids
1. To the aqueous solution of the extracts 10% ammonia solution is added
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and is heated. The production of fluorescence yellow is positive for
flavonoids.
2. 1ml of extract was taken and 10% of lead acetate was added. The yellow
precipitate is positive inference for the flavonoids
3. The extract is treated with concentrated H2SO4 resulting in the
formation of orange colour indicates the positive result for flavonoids.
4. To 5ml of dilute ammonia the plant extract is added and shaken well.
The aqueous portion is separated and concentrated H2SO4 is added. The
yellow colour indicates the presence of flavonoids.
Test for phytosterols
1. The extract is dissolved in 2ml of acetic anhydrite and to which 1 or 2
drops of concentrated H2SO4 is added along the sides an array of colour
change indicates the presence of phytosterols.
2. The extract was refluxed with alcoholic KOH and saponification takes
place. The solution was diluted with ether and the layer was evaporated and
the residue was tested for phytosterols. It was dissolved in diluted acetic
acid and few drops of concentrated H2SO4 are added. The presence of
bluish green colour indicates the presence of phytosterols.
Test for phlobatannins
1. Aqueous extract was boiled with diluted HCl leading to the deposition of
reddish precipitate indicates the presence of phlobatannins Test for
saponins 1. 0.5 mg of extract was vigorously shaken with few ml of distilled
water. The formation of frothing is positive for saponins
2. The froth from the above reaction is taken and few drops of olive oil is
added and shaken vigorously and observed for the formation of emulsion.
Test for steroids 2ml of extract with 2ml of chloroform and 2ml of
concentrated H2SO4 are added, the appearance of red colour and yellowish
green fluorescence indicates the presence of steroids
Test for xanthoproteins
1ml of extract is taken and to this few drops of nitric acid and
ammonia are added. Reddish brown precipitate indicates the presence of
xanthoproteins Test for chalcones 2ml of ammonium hydroxide is added to
0.5 g of extract. The appearance of red colour indicates the presence of
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chalcones
Test for Terpenoids (Salkowski test) 3ml of the extract was taken
and 1ml of chloroform and 1.5 ml of concentrated H2SO4 are added along
the sides of the tube. The reddish brown colour in the interface is
considered positive for the presence of terpenoids
Test for triterpenoids
To 10 mg of extract 1ml of chloroform is added and is mixed to
dissolve it. 2ml of concentrated H2SO4 is added followed by 1ml of acetic
anhydride. Formation of reddish violet colour is positive for the presence of
triterpenoids.
Test for anthocyanins
2ml of aqueous extract was taken to which 2N HCl was added and
it was followed by the addition of ammonia, the conversion of pink-red
turns blue-violet indicates the presence of anthocyanins.
Test for Leucoanthocyanins
To 5ml of extract dissolved in water, 5ml of Isoamyl alcohol is
added. The red appearance of the upper layer indicates the presence of
Leucoanthocyanins
Test for Coumarins
To 2 ml of the extract, 3 ml of 10% aqueous solution of NaOH is added.
The production of yellow colour indicates the presence of coumarins
Test for emodins
To 5ml of extract, 2ml of NH3OH and 3ml of benzene are added. The
production of red colour indicates the presence of emodins
Qualitative Analysis of Vitamins
Test for Vitamin – A
In 5 ml of chloroform, 250mg of the powdered sample is
dissolved and it is filtered, to the filtrate, 5ml of antimony trichloride
solution is added. The appearance of transient blue colour indicates
presence of vitamin-A
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Test for vitamin – C
In 5ml of distilled water, 1ml of the sample was diluted and a drop
of 5% sodium nitroprusside and 2ml of NaOH is added. Few drops of HCl
are added dropwise, the yellow colour turns blue. This indicates the
presence of vitamin- C
Test for vitamin – D
In 10 ml of chloroform, 500mg of powdered extract is dissolved
and filtered. 10ml of antimony trichloride is added, the appearance of
pinkish-red colour indicates the presence of vitamin – D
Test for vitamin – E
Ethanoic extract of the sample was made and filtered (500mg in
10ml), few drops of 0.1% ferric chloride were added and 1ml of 0.25% of
2’- 2’dipyridyl was added to 1ml of the filtrate. Bright-red colour was
formed with a white background.
Qualitative and Quantitative
Analysis Qualitative and quantitative analysis of phytochemicals
can be done using Gas Chromatography Mass Spectroscopy (GCMS).
GCMS can be applied to solid, liquid and gaseous samples. First the
samples are converted into gaseous state then analysis is carried out on the
basis of mass to charge ratio. High Performance Liquid Chromatography is
applicable for compounds soluble in solvents. High performance thin layer
chromatography is applicable for the separation, detection, qualitative and
quantitative analysis of phytochemicals.
Gas Chromatography
Volatile compounds are analysed using gas chromatography. In this
method, there is a gas and a liquid phase. The liquid phase is stationary
where the gas phase is a mobile phase. These compounds to be analysed are
also in the mobile phase with a carrier gas which is usually helium, hydrogen
or argon. The chemicals are separated depending on the migration rate into
the liquid phase. Higher percentage of the chemical will lead to faster
migration in the liquid phase. This is widely used in qualitative and
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quantitative phytochemical analysis.
High Performance Liquid Chromatography: (HPLC)
HPLC is also known as High- Pressure Liquid Chromatography.
This method involves the interaction of liquid solvent in the tightly packed
solid column or a liquid column. These acts as the stationary phase while
the liquid (solvent) acts as the mobile phase, high pressure enables the
compounds to pass to the detector. As HPLC compounds are analysed
after vaporisation, thermolabile compounds cannot be analysed with this
technique.
High Performance Thin Layer Chromatography: (HPTLC)
This method is modified form of thin layer chromatography. It is a
type of planer chromatography where the separation is done by high
performance layers with detection and the sample components are
acquisition using an advanced work- station. The reduction of the thickness
of the layer will increase the efficiency of the separation and hence HPTLC
is more advanced method for qualitative, quantitative and micro-preparative
chromatography.
Optimum Performance Laminar Chromatography:
(OPLC) OPLC combines the advantages of TLC and HPLC. The
system separates about 10-15 mg samples, with simultaneous processing of
up to 4 or 8 samples at a time depending on the model. In OPLC a pump is
used to force a liquid mobile phase through a stationary phase, such as silica
or a bonded-phase medium.
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